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Bioactivity Profile of Polyunsaturated Fattyacid extracts

from Sardinella longiceps and Sardinella fimbriata - A Comparative Study

Thesis submitted to the

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY In partial fulfilment of the requirements for the degree of

DOCTOR OF PHILOSOPHY In

MARINE BIOLOGY

Under the faculty of Marine Sciences

By

CHITRA SOM R.S

DEPT. OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY SCHOOL OF MARINE SCIENCES

COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY KOCHI – 682016

November – 2010

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Declaration

I hereby do declare that the thesis entitled “ Bioactivity Profile of Polyunsaturated Fattyacid extracts from Sardinella longiceps and Sardinella

fimbriata – A comparative study” is a genuine record of research work done

by me under the guidance of Dr. C.K. Radhakrishnan, Professor Emeritus, Department of Marine Biology, Microbiology and Biochemistry, Cochin University of Science and Technology and that no part thereof has been presented for the award of any degree, diploma, associateship, or other similar title of any University or Institution.

Kochi – 16 CHITRA SOM. R.S

November 2010

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I hereby declare that the thesis entitled “Bioactivity Profile of Polyunsaturated Fattyacid extracts from Sardinella longiceps and Sardinella fimbriata – A comparative study” is an authentic record of research work carried out by Ms. Chitra Som. R.S under my supervision in the School of Marine Sciences, Cochin University of Science and Technology in partial fulfilment of the requirements for the degree of Doctor of Philosophy and that no part thereof has been presented for the award of any degree, diploma or associateship in any University.

Dr. C. K. RADHAKRISHNAN

Professor Emeritus

Dept of Marine Biology, Microbiology and Biochemistry

School of Marine Sciences

Cochin University of Science and Technology Kochi - 682016

Kochi – 16

November 2010

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Acknowledgements

Carrying out the sheer volume and depth of this research was no easy task without the help of so many people and it is a pleasure to thank all those who made this thesis possible.

First and foremost, I am deeply grateful to my supervising guide Dr. C.K. Radhakrishnan, Retd. Professor, Department of Marine Biology, Microbiology

and Biochemistry, Cochin University of Science and Technology (CUSAT) whose encouragement, guidance and support from the initial to the final level enabled me to develop an understanding of the subject. He was generous in providing me this opportunity to receive my Ph.D. He has always been eager to help me through the toughest challenges during my research period.

I wish to express my sincere thanks to Dr. S. Yathiraj, Dean, Veterinary College, Bangalore (KVAFSU) for his valuable support, instructions and suggestions throughout my work period.

Sincere thanks are due to the Director and the Dean of School of Marine Sciences, Cochin University of Science and Technology.

I acknowledge with thanks the wholehearted support of all the Faculty of the Department of Marine Biology, Microbiology and Biochemistry. Thanks are due to the faculties who served as Department heads during the course of my Ph.D work. I am greateful to Dr. Anneykutty Joseph for the timely help she rendered. Sincere thanks are extended to Dr. Babu Philip and to Dr.Mohammed Hatha, on behalf of devoting their precious time for helping me with all the doubts I had about topics of their expertise.

Special Gratitude goes to my subject expert, Dr. Rosamma Philip and Dr A.V. Saramma,

for providing valuable suggestions and useful discussions. Thanks are due to Dr. Bijoy Nandan, for the help he rendered.

I acknowledge with thanks the help rendered by the Senior Scientists Dr. T.V. Sankar and Dr. R. Anandan of Central Institute of Fisheries Technology,

Kochi.

I owe my deepest gratitude to Dr. Lakshmi S and Prabha Pillai of Department of Molecular Medicine, Regional Cancer Center, Thiruvananthapuram for specific advice, guidance and help during work on cancer cell lines.

I would like to show my gratitude to the librarians of Cochin University of Science and Technology, Kerala University, Regional Cancer Center, Central Institute of Fisheries Technology, Central Marine Fisheries Research Institute, Indian Institute of

Science and National Centre for Biological Sciences. Special thanks are due to

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Dr. Tesmi Jose for providing me a visit to the library of National Oceanography Centre, Southampton, UK

My sincere thanks are due to all the office staff of the Department of Marine Biology, Microbiology and Biochemistry, CUSAT.

Expressing my gratitude to Abdul Jaleel, Nousher Khan and Jayanthi for their valuable assistance in the field. Without their help, the field work would not have been accomplished in time.

I appreciate the support and love rendered by Sini Wilson, Kiran, Meera, Dr. Anupama Nair, Dr. Bindhya Bhargavan and Dr.Sreedevi.

My special thanks are due to Dr. Gisha Sivan, Sreeja, Shamida, Nikitha, Roja Smitha, Naveen, Sapna, Afsal, Hari Shanker, Jisha, Remya, Smitha Bhanu, Sudha, Jini, Anil, Shyam, Cilla and my other fellow researchers in the Department of Marine Biology, Microbiology and Biochemistry.

I like to acknowledge the contributions of Anila Tony Mulloor for her editorial advice for this thesis.

I owe special gratitude to my parents for their continuous and unconditional support and for the motivation they gave me during those tiring times when I had doubts about my studies. Special thanks are due to my mom as she took the responsibility of taking care of my son in my absence. I am forever indebted to my parents–in-law for their understanding, endless patience and encouragement when it was most required.

My special gratitude is due to my sisters Jolly, Manju, Soorya and their families for their loving support.

I owe my loving thanks to my husband Praveen, the greatest blessing in my life, for being always there for me. Without all his love, help, encouragement, patience and understanding, it would have been impossible for me to finish this work. Thanks to my precious son, Tejas, for always soothing me with a smiling, loving face.

Finally, I would like to acknowledge all those who contributed in one way or

the other in helping to accomplish this PhD and the authors whose works formed the

basis upon which I built my research ideas. If I have unintentionally omitted to

recognise you, I apologise and accept the mistake as completely mine. God bless you all.

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Abstract

The oceans have proved to be an interminable source of new and effective drugs. Innumerable studies have proved that specific compounds isolated from marine organisms have great nutritional and pharmaceutical value. Polyunsaturated fattyacids (PUFA) in general are known for their dietary benefits in preventing and curing several critical ailments including Coronary heart disease (CHD) and cancers of various kinds.

Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) are two PUFA which are entirely marine in origin – and small Clupeoid fishes like sardines are known to be excellent sources of these two compounds. In this study, we selected two widely available Sardine species in the west coast, Sardinella longiceps and Sardinella fimbriata, for a comparative analysis of their bioactive properties. Both these sardines are known to be rich in EPA and DHA, however considerable seasonal variation in its PUFA content was expected and these variations studied. An extraction procedure to isolate PUFA at high purity levels was identified and the extracts obtained thus were studied for anti-bacterial, anti-diabetic and anti-cancerous properties.

Samples of both the sardines were collected from landing centre,

measured and their gut content analysed in four different months of the year

– viz. June, September, December and March. The fish samples were

analyzed for fattyacid using FAME method using gas chromatography to

identify the full range of fattyacids and their respective concentration in each

of the samples. The fattyacids were expressed in mg/g meat and later

converted to percentage values against total fatty acids and total PUFA

content. Fattyacids during winter season (Dec-Mar) were found to be

generally higher than spawning season (June-Sept). PUFA dominated the

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profiles of both species and average PUFA content was also higher during winter. However, it was found that S. longiceps had proportionately higher EPA as compared to S. fimbriata which was DHA rich. Percentage of EPA and DHA also varied across months for both species – the spawning season seemed to show higher EPA content in S. longiceps and higher DHA content in S. fimbriata. Gut content analysis indicate that adult S. fimbriata is partial to zooplanktons which are DHA rich while adult S. longiceps feed mainly on EPA rich phytoplankton. Juveniles of both species, found mainly in winter, had a gut content showing more mixed diet. This difference in the feeding pattern reflect clearly in their PUFA profile – adult S. longiceps, which dominate the catch during the spawn season, feeding mostly on phytoplankton is concentrated with EPA while the juveniles which are found mostly in the winter season has slightly less EPA proportion as compared to adults. The same is true for S. fimbriata adults that are caught mostly in the spawning season; being rich in DHA as they feed mainly on zooplankton while the juveniles caught during winter season has a relatively lower concentration of DHA in their total PUFA.

Various extraction procedures are known to obtain PUFA from fish

oil. However, most of them do not give high purity and do not use materials

indicated as safe. PUFA extracts have to be edible and should not have

harmful substances for applying on mice and human subjects. Some PUFA

extraction procedures, though pure and non-toxic, might induce cis-trans

conversions during the extraction process. This conversion destroys the

benefits of PUFA and at times is harmful to human body. A method free from

these limitations has been standardized for this study. Gas Chromatography

was performed on the extracts thus made to ensure that it is substantially

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pure. EPA: DHA ratios for both samples were derived - for S. longiceps this ratio was 3:2, while it was 3:8 for S. fimbriata.

Eight common strains of gram positive and gram negative bacterial strains were subjected to the PUFA extracts from both species dissolved in acetone solution using Agar Well Diffusion method. The activity was studied against an acetone control. At the end of incubation period, zones of inhibition were measured to estimate the activity. Minimum inhibitory concentration for each of the active combinations was calculated by keeping p < 0.01 as significant. Four of the bacteria including multi-resistant Staphylococcus aureus were shown to be inhibited by the fish extracts. It was also found that the extracts from S. fimbriata were better than the one from S.

longiceps in annihilating harmful bacteria.

Four groups of mice subjects were studied to evaluate the anti-

diabetic properties of the PUFA extracts. Three groups were induced

diabetes by administration of alloxan tetra hydrate. One group without

diabetes was kept as control and another with diabetes was kept as diabetic

control. For two diabetic groups, a prescribed amount of fish extracts were

fed from each of the extracts. The biochemical parameters like serum

glucose, total cholesterol, LDL & HDL cholesterol, triglycerides, urea and

creatinine were sampled from all four groups at regular intervals of 7 days

for a period of 28 days. It was found that groups fed with fish extracts had

marked improvement in the levels of total LDL & HDL cholesterol,

triglycerides and creatinine. Groups fed with extracts from S. fimbriata seem

to have fared better as compared to S. longiceps. However, both groups did

not show any marked improvement in blood glucose levels or levels of urea.

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Cell lines of MCF-7 (Breast Cancer) and DU-145 (Prostate Cancer) were used to analyse the cytotoxicity of the PUFA extracts. Both cell lines were subjected to MTT Assay and later the plates were read using an ELISA reader at a wavelength of 570nm. It was found that both extracts had significant cytotoxic effects against both cell lines and a peak cytotoxicity of 85-90% was apparent. IC

50

values were calculated from the graphs and it was found that S. longiceps extracts had a slightly lower IC

50

value indicating that it is toxic even at a lower concentration as compared to extracts from S. fimbriata.

This study summarizes the bioactivity profile of PUFA extracts and

provides recommendation for dietary intake; fish based nutritional industry

and indigenous pharmaceutical industry. Possible future directions of this

study are also elaborated.

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Contents

Page No

Chapter 1

Introduction ……….……….…….1-25

Review of Literature 5

1.1 Clupeoids and Sardines 5 1.1.1 Taxonomy 10 1.2 Marine Lipids and Polyunsaturated FattyAcids 13 1.2.1 Eicosapentaenoic Acid (EPA) 14 1.2.2 Docosahexaenoic acid (DHA) 15 1.2.3 Metabolism 15 1.3 Sardines and PUFA 17 1.4 PUFA and Nutrition 17

1.4.1 Pregnancy and Child Birth 19 1.4.2 Depression 19 1.4.3 Cardiac Disorders 20 1.4.4 Rheumatic Disorders 21 1.4.5 Cancer 21 1.4.6 Omega 3 Enriched Products 22 1.5 PUFA and Pharmaceuticals 22 1.6 Research Objectives 24

Chapter 2

Seasonal analysis of fattyacids in Sardinella

longiceps and Sardinella fimbriata ………..…………26-42

2.1 Introduction 26 2.2 Materials and Methods 27

2.2.1 Fish samples 27 2.2.2 Analysis of fatty acids 28 2.2.3 Extraction of Total Lipids 28 2.2.4 FattyAcid Methyl Ester Method 30 2.2.5 Presentation of Measures 31

2.3 Results 31

2.4 Discussion 38

2.5 Conclusion 42

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Chapter 3

Extraction of polyunsaturated fattyacids from

Sardinella longiceps and Sardinella fimbriata ………....43-50

3.1 Introduction 43 3.2 Materials and Methods 47

3.2.1 Preparation of the Extract 47 3.2.2 Saponification 47 3.2.3 Urea Complexing 48 3.2.4 Low temperature Fractional Crystallisation 48 3.3 Results and Discussion 49

3.4 Conclusion 50

Chapter 4

Anti-bacterial studies of PUFA extracts from

Sardinella longiceps and Sardinella fimbriata ………...…..51-63

4.1 Introduction 51 4.2 Materials and Methods 52

4.2.1 Extract Preparation and Determination of PUFA Composition 52 4.2.2 Antibacterial Assay 53 4.2.3 Statistical Analysis 54

4.3 Results 54

4.4 Discussion 60

4.5 Conclusion 63

Chapter 5

Anti-diabetic studies of PUFA extracts from

Sardinella longiceps and Sardinella fimbriata ………..……64-90

5.1 Introduction 64 5.1.1 Lipids and Diabetes 65 5.1.2 Kidney diseases and Diabetes 66 5.1.3 PUFA and Diabates 67 5.2 Materials and Methods 69

5.2.1 Extract Preparation and Determination of PUFA Composition 69

5.2.2 Experimental Animals 69

5.2.3 Induction of Experimental Diabetes 69

5.2.4 Study design 70

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5.2.5 Blood sampling 70 5.2.6 Analysis of Biochemcial Parameters 71 5.2.7 Statistical Procedure and Analysis 74

5.3 Results 74

5.3.1 Effects on Serum Glucose 76 5.3.2 Effect on Cholesterol and Triglycerides 76 5.3.3 Effects on Urea and Creatinine 82 5.3.4 GC Analysis 84

5.4 Discussion 84

5.4.1 Hypoglycemic Effect 85 5.4.2 Hypolipidemic Effects 86 5.4.3 Effects on Renal Functioning 88

5.5 Conclusion 90

Chapter 6

Anti-cancer studies of PUFA extracts from Sardinella longiceps and Sardinella fimbriata :

Breast and Prostate cancer………..…………..91-105

6.1 Introduction 91 6.1.1 Breast Cancer 92 6.1.2 Prostate Cancer 93 6.2 Materials and Methods 95

6.2.1 Extract Preparation and Determination of PUFA Composition 95 6.2.2 Cell Culture 95 6.2.3 Assessment of Cell Viability 96 6.2.4 Statistical Analysis 97

6.3 Results 97

6.4 Discussion 100

6.5 Conclusion 104

Chapter 7

Summary and Conclusion………..106-111

7.1 Recommendations for Dietary Intake 107 7.2 Recommendations for Nutrition Industry 109 7.3 Recommendations for Pharmaceutical Industry 110 7.4 Future Directions 110

APPENDIX

BIBLIOGRAPHY

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LIST OF FIGURES

Page No

Figure 1: EPA Structure 14

Figure 2: DHA Structure 15

Figure 3: Seasonal variation in fattyacid profile of Sardinella longiceps and Sardinela fimbriata 32 Figure 4: Seasonal variation of different types of fattyacids present in Sardinella longiceps 34 Figure 5: Seasonal variation of different types of fattyacids present in Sardinella fimbriata 34 Figure 6: Variations in EPA and DHA in Sardinella longiceps 35 Figure 7: Variations in EPA and DHA in Sardinella fimbriata 35 Figure 8: Variation in EPA concentration in Sardinella longiceps and Sardinela fimbriata 36 Figure 9: Variation in DHA concentrations of Sardinella longiceps and Sardinela fimbriata 36 Figure 10: Bray-Crutis similarity index for FA from Sardinella longiceps 37 Figure 11: Bray-Crutis similarity index for FA from Sardinella fimbriata 38 Figure 12: Comparison of antibacterial activity at highest concentration 57 Figure 13: Comparison of antibacterial activity at mid-concentration 58 Figure 14: Glucose variation in the four experimental groups 77 Figure 15: Urea variation in the four experimental groups 78 Figure 16: Total Cholesterol variation in the four experimental groups 78 Figure 17: Triglycerides variation in the four experimental groups 79 Figure 18: HDL Cholesterol variation in the four experimental groups 79 Figure 19: LDL Cholesterol variation in the four experimental groups 80 Figure 20: Recovery of Total Cholesterol in Sardinella longiceps and Sardinella fimbriata

extract treated groups 80

Figure 21: Recovery of Triglycerides in Sardinella longiceps and Sardinella fimbriata

extract treated groups 81

Figure 22: Recovery of LDL Cholesterol in Sardinella longiceps and Sardinella fimbriata

extract treated groups 81

Figure 23: Recovery of HDL Cholesterol in Sardinella longiceps and Sardinella fimbriata

extract treated groups 82

Figure 24: Creatinine Variation in the four experimental groups 83 Figure 25: Recovery of Creatinine in Sardinella longiceps and Sardinella fimbriata

extract treated groups 84

Figure 26: Cytotoxic activity of PUFA extracts on MCF-7 Cell line 99 Figure 27: Cytotoxic activity of PUFA extracts on DU-145 cell line 100

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LIST OF TABLES

Page No

Table 1: Sardines of the World 8

Table 2: Gas Liquid Chromatography Characteristics 30 Table 3: Sampling Details of Sardinella longiceps and Sardinella fimbriata 31 Table 4: Variation of Important Fattyacids in Sardinella longiceps and Sardinella fimbriata

across Seasons 33

Table 5: EPA and DHA content of Extract and Crude Oil 49 Table 6: Summary of Antibacterial activities of PUFA extract from Sardinella longiceps and

Sardinella fimbriata 54

Table 7: Antibacterial activity profile of Sardinella fimbriata. 55 Table 8: Antibacterial activity profile of Sardinella longiceps 56 Table 9: Minimum Inhibitory Concentration of PUFA extracts for Antibacterial activity 57 Table 10: Bio-chemical parameters analysed for all the sets 75 Table 11: Recovery in bio-chemical parameters in Sardinella longiceps and Sardinella fimbriata

extract treated groups 83

Table 12: Activity of PUFA extracts of Sardinella longiceps and Sardinella fimbriata on MCF-7

at different concentrations 98

Table 13: Activity of PUFA extracts of Sardinella longiceps and Sardinella fimbriata

extracts on DU-145 at different concentrations 98 Table 14: Summary of the cytotoxic activity of PUFA extracts on MCF -7 and DU-145 cell lines 99 Table 15: Summary of Studies on the two Sardine Extracts 108

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ABBREVIATIONS

Acronym Expansion AA Arachidonic Acid

ALA Alpha-Linolenic Acid AOAC

Association of Analytical Communities - known as AOAC International now

AOE Anti-Oxidant Enzyme CHD Coronary Heart Disease

COX Cyclooxygenase. An enzyme in human body

CPT-11 Irinotecan. A cytotoxic quinoline alkaloid used in treatment of cancer CVD Cardio-Vascular Disease

DHA DocosaHexaenoic Acid DISC Death-Inducing Signalling Complex DMEM Dulbecco’s Modified Eagle Medium DU-145 A human prostate cancer cell line EFA Essential Fatty Acid

EPA EicosaPentaeonic Acid FA FattyAcid

FAME Fatty Acid Methyl Ether - known as FAME Method GC Gas Chromatography

HDL High-density Lipoprotein - known as HDL Cholesterol hK2 Hexokinase 2. An enzyme in human body

IC50 Half maximal inhibitory concentration

LDL Low-density Lipoprotein - known as LDL Cholesterol LOX Lipooxygenase. An enzyme in human body

MCF-7 A human breast cancer cell line MDA-MB A set of human breast cancer cell lines

MHA Mueller Hinton Agar – known as MHA medium MIC Minimum Inhibitory Concentration MRA Magnetic Resonance Angiogram

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MRSA Methicillin-Resistant Staphylococcus aureus

MTT 3- (4, 5- dimethylthiazol-2yl)-2, 5- diphenyltetrazolium bromide) MUFA Mono-Unsaturated FattyAcid

MX-1 A human breast cancer cell line

NSAID Non-Steroidal Anti-Inflammatory Drugs PSA Prostate Specific Antigen

PUFA Poly-Unsaturated FattyAcid ROS Reactive Oxygen Species

SEM Scanning Electron Microscopy SFA Saturated FattyAcid

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Chapter 1

Introduction

Review of Literature 1.1 Clupeoids and Sardines

1.1.1 Taxonomy

1.2 Marine Lipids and Polyunsaturated Fattyacids 1.2.1 Eicosapentaenoic Acid (EPA)

1.2.2 Docosahexaenoic acid (DHA)

1.2.3 Metabolism 1.3 Sardines and PUFA 1.4 PUFA and Nutrition

1.4.1 Pregnancy and Child Birth 1.4.2 Depression

1.4.3 Cardiac Disorders 1.4.4 Rheumatic Disorders 1.4.5 Cancer

1.4.6 Omega 3 Enriched Products 1.5 PUFA and Pharmaceuticals 1.6 Research Objectives

The issues that plague world health have swerved with the times.

New pharmaceuticals drugs, research into the lifecycle of pathogens, disease prevention and vaccination have helped eradicate major health risks posed by infections, and many diseases are believed to be eradicated. The report of World Heath Organisation (WHO) on global patterns of health risks renders plenty of evidence that the major health risks we face today do not lie with any particular infection but are the consequences of an unhealthy lifestyle and perverted eating habits.

C o n t e n t s

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Chapter 1

Introduction

2

As stated in the report of WHO, the top five contributors to world’s health problems are high blood pressure, tobacco use, high blood glucose, physical inactivity, overweight and obesity. Next up on the list is high cholesterol. These factors are responsible for raising the risk of chronic diseases, such as heart disease and cancers. They affect countries across all income groups: high, middle and low. Internationally, health experts are now asserting the importance of a healthy lifestyle that involves a conscientious diet and meticulous exercise regimen. ‘Prevention is better than cure’ as the saying goes.

The food that traditionally forms the staple of the local diet goes far in ensuring the health of a population - preventing the onslaught of the said maladies, and exerting a positive influence where they have made their ominous presence already felt. It is no secret that the longevity and wellness enjoyed by the Japanese, particularly Okinawans, are a result of their centuries-old dietary habits which include plenty of seaweed and fish. On an average, a Japan national consumes half a pound of fish each day. Japanese women also command the lowest rate of obesity in modern cultures.

Another popular model that illustrates the extent to which diet

influences the health of a popular is the diet of Alaskan Eskimos. These

communities that survive in some of the most strenuous climates on the

planet and eat a diet that is nearly 70% fats still manage to astonish their

western meat-eating counterparts with an extremely low incidence of

cardiac illnesses and also joint and skin diseases. Research reveals that their

diet includes lot of fish, rich in the two major ω-3 Fatty Acids -

Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA).

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Chapter 1

Introduction

The role of these essential fatty acids in promoting cardiac health stems from the fact that they can improve the performance of heart and bring down cholesterol levels and blood pressure. And in so doing they protect us against Cardiovascular Diseases (CVD). One cannot overemphasize the import of this in healthcare: according to WHO Fact Sheets CVDs is the number one killer in the world accounting for 29% (17.1 million) deaths globally in 2004. By lowering blood pressure in hypertensive individuals PUFAs can axe almost half of all strokes and ischemic heart diseases. The physical inactivity brought about by our lifestyle and nature of employment is the fourth most threatening health risk as it causes around 21–25% of breast and colon cancer. It is a well known fact that PUFAs can lower the risk of cancer and assist in its treatment. So their presence in our diet can go a long way in eliminating the risk of cancer, and treatments can be augmented by an inclusion of PUFA supplements as well.

PUFAs are also essential for the proper development of brain. A markedly positive influence of these bio-molecules has been noted on neurological disorders like Attention Deficit Hyperactive Disorders in Children, brain disorders like Alzheimer’s and mental illnesses like depression and bipolar disorder. Researchers are also optimistic about the assistance PUFAs can provide in the treatment of diseases like lupus and psoriasis.

PUFAs are absolutely essential to the body in performing its cellular

functions effortlessly. Cell to cell signalling and biochemical functions at

molecular levels are mediated and perfected by the presence of these fatty

acids in our body. They also form precursors of substances with hormonal

regulator properties. Bolstering our immune system, both EPA and DHA

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Chapter 1

Introduction

4

help to check abnormal immune responses in asthma, rheumatoid arthritis, kidney inflammation and eczema. Both gestating and lactating mothers and their babies can benefit from an inclusion of these fatty acids in the diet, which helps proper brain development and good vision.

Fish, being high in PUFA content and low in harmful cholesterol, is an ideal source of EPA and DHA for regular consumption. Fishes vary in the abundance of these molecules. If one is hoping to include more omega 3 fatty acids in the diet the best choices are sardines, mackerel, salmon, herring and menhaden. Sardines, are one of the most abundant and cheaply available fishes across the Indian coast line, has long been an integral part of the diet in Kerala, Coastal Karnataka and Tamil Nadu.

With many countries of the Indian Ocean belt – almost 500 million people - face a serious deficiency of proteins. Sardines can be utilized as an affordable source of excellent proteins. Sardine fishery, if put to proper use, and underpinned by spreading awareness, can bring down underweight in children, which is the major global contributor to increasing Disability- Adjusted Life Years (DALY) - the number of years lost due to ill-health, disability or early death. Sardines have now achieved added significance because of the discovery that it is a fish exceptionally rich in EPA and DHA. A regular inclusion of the fish in the diet can contribute a great deal to healthcare and help us grapple more effectively with cardiac illnesses, diabetes and cancer that affect a large segment of our population.

A steady supply of fresh sardines is possible only across our coastal belt.

For the benefit of inland populations an effective processing method can be

developed which will ensure that they get the valuable components in the

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Chapter 1

Introduction

fish intact. This will also guarantee a longer shelf life and perennial availability.

In keeping with the trend of the day, brands of everyday food such as fruit juices, breads, spreads, margarines, snack bars, yogurt, fish products, eggs and children’s beverages enriched with DHA and EPA have invaded market shelves. It is a quick and effective method to deliver the necessary fatty acids to the body, even for strict vegetarians. Trials to produce PUFA- enriched eggs by feeding marine PUFA to egg laying hens have yielded encouraging results. Even fast food like hot dogs and frankfurters can be absolved from the infamy of ‘greasy foods’ by enriching them with PUFA.

It helps people to eat healthy even when they are on the move.

Review of literature

1.1 Clupeoids and Sardines

Sardines are a group of small fishes classified under three genus;

Sardina, Sardinops and Sardinella; together consisting of 23 species

(Table 1) under the widely categorized group of fishes known as Clupeoids.

Clupeoid fishes are small teleosts which are relatively ‘unevolved’ and

typically are less than 20 or 30 cm in length; they have no barbules and are

frequently laterally compressed, with a series of hard scutes along the

ventral surface of the body. Most are soft-bodied, difficult to handle, and

covered with rather deciduous scales. Their flesh is often very oily, and the

oil content normally varies seasonally, even in low latitude species

(Longhurst 1971). These fishes are commercially extremely important – in

fact clupeoid fishes like Herrings, Pilchards and Sardines form the main stay

of economy of the European maritime nations – as aptly coined by the

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Chapter 1

Introduction

6

famous French expression ‘la crise sardinere’ refering to the disastrous effect of the failed sardine fishery and its impact on the nation. While only a single genus (Clupea) of clupeoids is important in high latitudes and the cold temperate regions, in the warm temperate mid-latitudes there are approximately 10 important genera, dominated by Sardina, Sardinops,

Engraulis, Brevoortia, Etlzmidium and Opisthonema. In low latitudes more

than 25 genera occur, many of which are important of which Sardinella being the prime (Longhurst 1971).

One of the most significant characteristics of clupeoids from a behavioural and fisheries viewpoint is their occurrence in dense and often very large schools containing many hundreds of thousands of fish which may weigh up to more than 100 tons (Longhurst 1971). Such large schools generally occur in the open ocean, particularly for pelagic sardines and frequently have a diagnostic shape and shoaling behaviour which enabled experienced fishermen to identify them. Clupeiformes also congregate in smaller, less-organized shoals, particularly during spawning seasons. In addition to schooling, some clupeoid fishes may migrate inshore or across latitudes on a seasonal basis (Longhurst 1971).

Most Clupeiformes filter feed by straining water through their long

and numerous gill rakers. They consume plankton, particularly small

crustaceans and the larval stages of larger crustaceans and fishes. The

species which have a diet in which phytoplankton appears to be the

preferred component, feed by filtering large diatoms and other

phytoplanktons from the water by means of elongated gill rakers which

form a filtration sieve. Though few in number, from a fisheries point of

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Chapter 1

Introduction

view these species are some of the most interesting, both because of their occurrence in great quantity and also because they represent the most efficient possible utilization of the results of primary production. Indian Oil- sardine

Sardinella longiceps (Peterson 1956;

Nair 1960) fall into this category.

The Indo-Pacific region also contains the greatest number and

diversity of species of clupeoids in the tropical oceans; along the mainland

of Asia from China to the Red Sea, down the coast of eastern Africa, and

through the Indonesian, the Philippine, and the Australian archipelagos. In

Indian waters, the clupeoids are chiefly represented by Sardines, Anchovies,

White-taits among which Indian Oil Sadrine (Sadinella longiceps) ranks as

the most valuable and forms the backbone of the fishery of the west coast of

India. At least three other species of Sardines can also be found in the south-

west coast of India where this study was conducted viz. Sardinella

fimbriata, Sardinella gibbosa, Sardinella jussieu; listed in decreasing order

of abundance. The present study concentrates on two widely available

Sardines in this area – S. longiceps and S. fimbriata and attempts to compare

their fatty acid profile with their corresponding variations in bioactivity.

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8

Sardines of the World

Table 1: Sardines of the World Common Name Scientific Name Distribution

European Pilchard Sardina pilchardus

NE Atlantic: Iceland (rare) and N. Sea, southward to Bay de Gorée, Senegal.

Mediterranean (common in the W. part and in Adriatic Sea, rare in the E. part), Sea of Marmara and Black Sea

South American

Pilchard Sardinops sagax

Indo-Pacific: S. Africa to the E. Pacific. Three lineages: S. Africa (ocellatus) and Australia (neopilchardus), Chile (sagax) and California (caeruleus) and Japan (melanostictus) White Sardinella Sardinella

albella

Indo-West Pacific: Red Sea, Persian Gulf, E.

African coasts, Madagascar E. to Indonesia and the Arafura Sea, N. to Taiwan and S. to Papua New Guinea.

Bleeker's blacktip

Sardinella Sardinella

atricauda W. Pacific: Indonesia

Round Sardinella Sardinella aurita

E. Atlantic: Gibraltar to Saldanha Bay, S.

Africa. Also known from the Mediterranean and Black Sea. W. Atlantic: Cape Cod, USA to Argentina. Bahamas, Antilles, Gulf of Mexico and Caribbean coast

Deepbody

Sardinella Sardinella brachysoma

Indo-West Pacific: Madagascar (but apparently not elsewhere in the W. Indian Ocean), Madras, Indonesia, N. Australia

Fiji Sardinella Sardinella fijiense

W. Pacific: Papua New Guinea and Fiji.

Reported from New Caledonia Fringescale

Sardinella Sardinella fimbriata

Indo-West Pacific: S. India and Bay of Bengal to the Philippines, also E. tip of Papua New Guinea

Goldstripe Sardinella

Sardinella gibbosa

Indo-West Pacific: Persian Gulf, East Africa and Madagascar to Indonesia, north to Taiwan and Korea south to the Arafura Sea and northern Australia.

Taiwan Sardinella Sardinella

hualiensis NW Pacific: Taiwan, possibly S. to Hong Kong Brazilian

Sardinella Sardinella

janeiro W. Atlantic: Gulf of Mexico, Caribbean, W.

Indies S. to Brazil and N. Uruguay Mauritian

Sardinella Sardinella jussieu

W. Indian Ocean: W. coasts of S. India, from Bombay S. to Sri Lanka; also to Madagascar and Mauritius. NW Pacific: Taiwan, Hong Kong and Viet Nam

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Bali Sardinella Sardinella lemuru

E. Indian Ocean: Phuket, Thailand; southern coasts of E. Java and Bali; and W. Australia. W.

Pacific: Java Sea, Philippines, Hong Kong, Taiwan Island, S. Japan

Indian Oil Sardine Sardinella longiceps

Indian Ocean: N. and W. parts only, Gulf of Aden, Gulf of Oman, but apparently not Red Sea or the Persian Gulf, E. to S. part of India, on E. coast to Andhra; possibly to the Andaman Islands.

Madeiran

Sardinella Sardinella maderensis

E. Atlantic: Gibraltar to Angola; single specimen recorded from Walvis Bay, Namibia.

Also known from the Mediterranean (S. and E.

parts, also penetrating Suez Canal).

Marquesan

Sardinella Sardinella

marquesensis E. Pacific: endemic to the Marquesan Islands.

Introduced into Hawaiian waters.

Blacktip Sardinella Sardinella melanura

Indo-West Pacific: Gulf of Aden S. to

Madagascar and Mauritius and E. to the Arabian Sea and NW India (apparently not found S. of Bombay nor in N. Bay of Bengal); then from Indonesia (but not in S. China Sea) to Samoa.

Reported from the Penghu Islands

East African Sardinella

Sardinella neglecta

W. Indian Ocean: Somalia, Kenya, and Tanzania

Richardson's Sardinella

Sardinella

richardsoni NW Pacific: Hainan Island, Hong Kong, China.

Yellowtail

Sardinella Sardinella rouxi E. Atlantic: Senegal to Congo and perhaps S. of Angola.

Sind Sardinella Sardinella

sindensis W. Indian Ocean: Arabian Sea, from Gulf of Aden to the Persian Gulf and Bombay Freshwater

Sardinella Sardinella

tawilis Endemic to Lake Taal (= Lake Bombon), Luzon, Philippines

Japanese Sardinella

Sardinella zunasi

W. Pacific: S. coasts of Japan S. to about Taiwan

Source: www.fishbase.org

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10 1.1.1 Taxonomy

Taxonomic positions of the two test species,

Sardinella longiceps

and Sardinella fimbriata are given below

Kingdom Animalia

¾ Phylum Chordata

¾ Class Actinopterygii

¾ Order Clupeiformes

¾ Family Clupeidae

¾ Genus Sardinella

¾ Sardinella longiceps (Valenciennes, 1847)

¾ Sardinella fimbriata (Valenciennes, 1847)

Indian Oil-Sardine - Sardinella longiceps

Fringescale Sardine - Sardinella fimbriata

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Sardinella longiceps is identified by its sub-cylindrical elongated

body with its ventral profile evenly convex. It can be conclusively separated from all other clupeids in the northern Indian Ocean by its longer head and lower gill rakers. Caudal fin is well forked, lobes pointed; two large alar scales can be seen at the base, colour bluish green back with golden reflections, abdomen silvery with pinkish tinge and a faint golden spot behind gill opening are other in-hand diagnosis (Whitehead 1985).

Sardinella fimbriata can be identified by its compressed, flattened

body and conclusively by its total number of scutes which varies consistently from 29 to 33. Vertical striae on scales do not meet at center, hind part of scales have a few perforations and somewhat produced posteriorly. A dark spot at dorsal fin origin also can be seen (Whitehead 1985).

Sardinella longiceps is an extremely valuable commercial fish and is

also the most important clupeoid fishery of the whole of western Indo- Pacific. Stocks of this species extend around the whole perimeter of the northern part of the Indian Ocean from the Gulf of Aden to the Bay of Bengal and also occur in the Indonesian archipelago and the Philippines.

Malabar region is considered as the zone of maximum abundance. The landings of this important species in India have reached as much as 200 thousand tons per annum in some years (Mohanty et. al.). During the Second World War, people of Kerala purely sustained themselves on the traditional dish of sardine and tapioca to save them from the bitter famine.

However, the fishery is susceptible to irregular and large-scale fluctuations

in resource availability and hence it has been studied intensively by fishery

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12

biologists in India, beginning in the early 1920s with the work of Hornell and his associates. When Central Marine Fisheries Research Institute (CMFRI) was established in 1947, its top priority area was to study the Indian Oil Sardine. Since then, investigations on systematics, fishery, food and feeding, growth, distribution, reproduction, nutritional value and processing were extensively carried out which enriched our knowledge on this species. The shoals of Indian Oil Sardines become available to the fishery towards the end of June, when populations of adults with mature gonads appear near the coast and progressively move northwards as the season advances; these fish have mature gonads and spawn during their first few months in the coastal region. As the season advances, a second wave of shoals arrives in the coastal region and becomes available to the fishery;

these are younger, immature fish and their availability reaches a peak during

the months of October to December. Approximately the same cycle of

events is repeated annually on the east coast of India. The arrival of the first

wave of adult sardines at the coast generally coincides with the onset of the

southwest monsoon; at this time there is a very strong seasonal bloom of

phytoplankton, principally of the diatom Fragillaria sp. and it is supposed

that the spawning migration is timed to coincide with this; the arrival of the

second wave of such shoals, at a peak in October to December coincides

with a second phytoplankton bloom. Nair (1960) has shown that the

stomach contents of this species are dominated by phytoplankton, largely a

single species of the diatom (Fragillaria oceanica) and dinoflagellates. He

has suggested that the large and important fluctuations in the availability of

the species from year to year may be dependent upon the nature and timing

of the annual bloom of this diatom.

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S. fimbriata is comparatively a less important lesser Sardine in terms

of the trade and the species has an average annual landing of around 50000 tonnes in India (Mohanty et al.). This species is found in the local fisheries of Philippines, in the Visayan Sea in Indonesia, along the south-east coast of Bay of Bengal and southern coast of the Arabian Sea, and along the north coast of the Australian continent. This species occurs in commercially significant quantities in the southern part of Indian coast. These are mostly zooplankton-feeders and where their diet has been investigated (Ronquillo 1960) it is evident that they subsist upon a mixed diet, dominated by crustacea of various sorts, according to the relative availability from place to place and season to season. Spawning season extends from August to February (Bennet 1965) with juveniles appearing in the catch almost at the same time as S. longiceps.

1.2 Marine Lipids and Polyunsaturated Fattyacids

Marine lipids from sardines come under two categories of fattyacids;

Saturated Fattyacids (SFA) and Unsaturated Fattyacids (USFA).

Unsaturated Fattyacids are characterized by the presence of at least one

double bond between the carbons. Unsaturated fattyacids consist of

monounsaturated fattyacids (MUFA) and polyunsaturated fattyacids

(PUFA). There are two classes of PUFAs, ω3 and ω6. The distinction

between

ω3 and ω6 fattyacids is based on the location of the first double

bond, counting from the methyl end of the fattyacid molecule. ω3 and ω6

fattyacids are also known as essential fattyacids (EFAs) because humans,

like all mammals, cannot make them and must obtain them in their diet

(Bendich and Deckelbaum 2005).

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14

The base origin of most marine lipids are from phytoplankton which are rich in PUFA(Klein Breteler et al. 1999). However, zooplanktons are also known to assimilate PUFA with higher levels of unsaturation. This extra level of unsaturation is partly achieved in zooplankton from their microplankton based diet. Microplanktons are known to preferentially increase the level of unsaturation in the food chain by converting the PUFA obtained from phytoplankton to higher degrees of unsaturation (Kleppel

et al. 1998, Klein Breteler et al. 1999). Two important naturally occurring ω3 fatty acids that are entirely marine based, are Eicosapentaenoic Acid

(EPA) and Docosahexaenoic Acid (DHA) (Klein Breteler et al. 1999). EPA and DHA are essential as structural components of all the cell walls. They are necessary for proper brain and eye development, and are required for the proper functioning of the immune, reproductive, respiratory and circulatory systems (Simopoulos 1991).

1.2.1 Eicosapentaenoic Acid (EPA)

EPA, systematically called all-cis-5, 8, 11, 14, 17-icosapentaenoic acid (Figure 1), is a carboxylic acid with a 20-carbon chain with five cis- double bonds (sometimes denoted as C20:5(n-3)). This FA is involved in the production of eicosanoids, which are hormone-like substances which act as vasodilators and anti-platelet aggregators. EPA is a precursor to the eicosanoids known as series 3 prostaglandins and thromboxanes and series 5 leukotrienes (Arthur 1999).

Figure 1: EPA Structure

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1.2.2 Docosahexaenoic acid (DHA)

DHA, systematically called as all-cis-docosa-4,7,10,13,16,19-hexa- enoic acid (Figure 2), is a carboxylic acid with a 22-carbon chain and six cis double bonds (denoted sometimes as C22:6(n-3)). DHA is an important component of our brain and eyes. It is fundamentally important in the neurological growth and development of children, and for their eyesight (Arthur 1999).

Figure 2: DHA Structure

1.2.3 Metabolism

Omega 6 fatty acids are represented by linoleic acid (LA) and the

corresponding

ω3 fatty acids by ά-linolenic acid (ALA). LA is plentiful in

nature and is found in the seeds of most plants except for coconut, cocoa,

and palm. ALA on the other hand is found in the chloroplast of green leafy

vegetables. Both EFAs can be metabolized to longer-chain fatty acids of 20

and 22 carbon atoms. LA is metabolized to arachidonic acid (AA) and ALA,

to EPA and DHA, increasing the chain length and degree of unsaturation by

adding extra double bonds to the carboxyl group. Humans and animals

except carnivores such as lions and cats can convert LA to AA and ALA to

EPA and DHA (de Gomez & Brenner 1975). There is competition between

ω3 and ω6 fattyacids for the desaturation enzymes. However, both 1-4 and

1-6 desaturase prefer ω3 to ω6 fattyacids. There is some evidence that 1-6

desaturase decrease with age. Premature infants, hypertensive individuals,

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16

and some diabetics are limited in their ability to make EPA and DHA from ALA. These findings are important and the role of fish oils which are natural sources of EPA and DHA is quite significant in these cases (Simopoulos 1991). AA is found predominantly in the phospholipids of grain-fed animals. LA, ALA, and their long-chain derivatives are important components of animal and plant cell membranes. In mammals and birds the

ω3 fattyacids are distributed selectively among lipid classes. ALA is found

in triglycerides, in cholesteryl esters, and in very small amounts in phospholipids. EPA is found in cholesteryl esters, triglycerides, and phospholipids. DHA is found mostly in phospholipids. In mammals, including humans, the cerebral cortex, retina, and testis and sperm are particularly rich in DHA. DHA is one of the most abundant components of the brain’s structural lipids. DHA, like EPA, can be derived only from direct ingestion or by synthesis from dietary EPA or ALA (Simopoulos 1991).

It has been reported that conversion of ALA to EPA and further to DHA in humans is limited, but varies with individuals. Women have higher ALA conversion efficiency than men, probably due to the lower rate of utilization of dietary ALA for beta-oxidation. This suggests that biological engineering of ALA conversion efficiency is possible (Hussein et al. 2005).

Goyens et al. (2006) suggest that it is the absolute amount of ALA, rather than the ratio of

n−3 and n−6 fattyacids, which affects the conversion.

However, ALA-feeding studies and stable-isotope studies using ALA,

which have addressed the question of bioconversion of ALA to EPA and

DHA, have concluded that in adult men conversion to EPA is limited

(approximately 8%) and conversion to DHA is extremely low (<0.1%). In

women fractional conversion to DHA appears to be greater (9%), which

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may partly be a result of a lower rate of utilisation of ALA for beta- oxidation in women. In this context, direct intake of sufficient quantities of these ω3 FA is essential for the stable metabolism of the body (Simopoulos 1991).

1.3 Sardines and PUFA

Clupeid fishes are known to be seasonal feeders. They store great reserves of energy for maintenance during the times when food is scarce.

Fishes store their energy as lipids and these compounds are burnt when energy has to be expended. They are also useful in maintaining stability, permeability and fluidity of the cell membranes. Fat reserves and fattyacid composition of the fishes can vary with age, sex and season. This has been proved in several species of clupeids including S. longiceps (Gopakumar 1965) apart from other Sardinops (Gamez-Mezza et al. 1999, Shirai et al.

2002) and Sardina (Bandarra et al. 1997).

Easy availability of these species in the western coast in great quantities roughly all through out the year means a ready availability of enormous amounts of these essential FAs for human consumption. This brings in a huge commercial implication for the fishery industry as this can potentially supplement a viable nutritional and pharmaceutical industry solely based on Marine PUFA.

1.4 PUFA and Nutrition

Scientists were first alerted to the many benefits of fish oils in the

early 1970s when Danish physicians observed that Greenland Eskimos had

an exceptionally low incidence of heart disease and arthritis despite the fact

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18

that they consumed a high-fat diet. Intensive research soon discovered the secret that two of the fats (oils) they consumed in large quantities, EPA and DHA, were actually highly beneficial. More recent research has established that fish oils (EPA and DHA) play a crucial role in the prevention of atherosclerosis, heart attack, depression, and cancer.

Seemingly minor differences in their molecular structure make the two EFA families act very differently in the body. While the metabolic products of ω6 FA promote inflammation, blood clotting, and tumor growth, the ω3 FA act entirely opposite (Caygill et al. 1996). Although both ω3s and

ω6s are needed, it is becoming increasingly clear that an excess of ω6 FA

can have dire consequences. Many scientists believe that a major reason for the high incidence of heart disease, hypertension, diabetes, obesity, premature aging, and some forms of cancer is the profound imbalance between our intake of ω6 and ω3 FAs. Our ancestors evolved on a diet with a ratio of ω6 to ω3 of about 1:1. A massive change in dietary habits over the last few centuries has changed this ratio to something closer to 20:1 and this spells trouble (Simopoulos 1991).

Recognizing the unique benefits of EPA and DHA and the serious

consequences of a deficiency the US National Institutes of Health recently

published Recommended Daily Intakes of fattyacids. It recommends a total

daily intake of 650 mg of EPA and DHA, 2.22 g/day of ALA and 4.44 g/day

of LA. Saturated fat intake should not exceed 8% of total calorie intake or

about 18 g/day.

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1.4.1 Pregnancy & Child Birth

An adequate intake of DHA and EPA is particularly important during pregnancy and lactation. During this time the mother must supply all the baby's needs because it is unable to synthesize these essential fattyacids itself. DHA makes up 15 to 20% of the cerebral cortex and 30 to 60% of the retina (Gal et al. 2005). There is some evidence that an insufficient intake of

ω3 fattyacids may increase the risk of premature birth and an abnormally

low birth weight (Carlson 1999, Cunnane et al. 2000, Makrides et al. 2000).

There is also emerging evidence that low levels of omega-3 acids are associated with hyperactivity in children (Mitchel et al. 1987). The constant drain on a mother's DHA reserves can easily lead to a deficiency and it is believed that pre-eclampsia and postpartum depression could be linked to a DHA deficiency. Experts recommend that women get at least 500-600 mg of DHA every day during pregnancy and lactation (Carlson 1999).

1.4.2 Depression

The human brain is one of the largest "consumers" of DHA. A normal adult human brain contains more than 20 grams of DHA. Low DHA levels have been linked to low brain serotonin levels which again are connected to an increased tendency to depression, suicide, and violence (Edwards

et al. 1998). Studies have shown that countries with a high level

of fish consumption have fewer cases of depression (Hibbeln 1998).

Researchers at Harvard Medical School have successfully used fish oil

supplementation to treat bipolar disorder (Stoll et al. 1999) and British

researchers report encouraging results in the treatment of schizophrenia

(Laugharne et al. 1996).

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20 1.4.3 Cardiac Disorders

Danish researchers have concluded that fish oil supplementation

may help prevent arrhythmias and sudden cardiac death in healthy men

(Christensen

et al. 1999). An Italian study of 11,000 heart attack survivors

found that patients supplementing with fish oils markedly reduced their risk

of another heart attack, a stroke or death. A group of German researchers

found that fish oil supplementation for two years caused regression of

atherosclerotic deposits (von Schacky et al. 1999) and American medical

researchers report that men who consume fish once or more every week

have a 50% lower risk of dying from a sudden cardiac event than do men

who eat fish less than once a month (Siscovick et al. 1995). Fish oil

supplementation (10 grams/day) reduces the number of attacks by 41% in

men suffering from angina (Salachas et al. 1994). It is found that fish oil

supplementation reduces the severity of a heart attack and supplementation

started immediately after a heart attack reduces future complications

(Eritsland

et al. 1994). Bypass surgery and angioplasty patients reportedly

also benefit from fish oils and clinical trials have shown that fish oils are

beneficial for heart disease patients (Singh et al. 1997). Fish oils are

especially important for diabetics who have an increased risk of heart

disease. It is found that supplementing with as little as 2 grams/day of fish

oil (410 mg of EPA plus 285 mg of DHA) can lower diastolic pressure by

4.4 mm Hg and systolic pressure by 6.5 mm Hg in people with elevated

blood pressure (Appel et al. 1993).

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1.4.4 Rheumatic Disorders

Fish oils are particularly effective in reducing inflammation and can be of great benefit to people suffering from rheumatoid arthritis or ulcerative colitis. Daily supplementation with as little as 2.7 grams of EPA and 1.8 grams of DHA can markedly reduce the number of tender joints and increase the time before fatigue sets in (Kremer 2000). Some studies have also noted a decrease in morning stiffness (Fortin et al. 1995) and clinical trials concluded that arthritis patients who took fish oils could eliminate or sharply reduce their use of NSAIDs and other arthritis drugs (Kremer et al.

1995).

1.4.5 Cancer

There is also considerable evidence that fish oil consumption can reduce the risk of breast and prostate cancer (Chavarro et al. 2008) and help slow their progression (Caygill et al. 1996). Daily supplementation with fish oils has been found effective in preventing the development of colon cancer (Mehta

et al. 2008). There is now also considerable evidence that fish oil

consumption can delay or reduce tumor development in breast cancer.

Studies have also shown that a high blood level of omega-3 fattyacids

combined with a low level of ω6 acids reduces the risk of developing breast

cancer (Soto-Guzman et al. 2010). Daily supplementation with as little as

2.5 grams of fish oils has been found effective in preventing the progression

from benign polyps to colon cancer (Fernandez-Banares et al. 1996) and

Korean researchers reported that prostate cancer patients have low blood

levels of omega-3 fattyacids (Yang et al. 1999). Greek researchers report

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Chapter 1

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22

that fish oil supplementation improves survival and quality of life in terminally ill cancer patients (Gogos 1998).

1.4.6 Omega 3 Enriched Products

Omega 3 fattyacids are being increasingly promoted as important dietary components for health and disease prevention. These fattyacids are naturally enriched in fatty fish like salmon and tuna and in fish-oil supplements. An increasing number of foods that are not traditional sources of ω3 fattyacids, such as dairy and bakery products, are now being fortified with small amounts of these fattyacids (Surette 2008).

In conclusion, the direct and indirect nutritional advantages of ω3 fattyacids have been recognised by the medical community and increasing presence of ω3 enriched food products is a testimony to this fact.

1.5 PUFA and Pharmaceuticals

Omega 3 oils, though called ‘miracle food’ of the 21

st

century, are not a ‘miracle drug’ in itself. Its use in pharmaceutical industry is always in combination with a more direct drug and the presence of these FA induce a favourable condition in the patient’s body for the real drug to be effective.

FA in combination with drugs for the treatment of diseases is an area

of immense interest because it opens a new field in pharmaceutical research

-

ω3 fattyacids in the control of metabolic and autoimmune disorders, that

includes CVD, arthritis, nephrites, psoriasis, ulcerative colitis and cancer

(Simopoulos et al. 1991). Preliminary data from animal and human studies

suggest that the concurrent ingestion or administration of ω3 fattyacids with

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drugs leads to potentiation of drug effects, as with propranolol, which may lead to a decrease both in the dose of ω3 fattyacids and in the drug dose or, as with cyclosporine, to a decrease in toxicity of the drug. By partially replacing the fattyacids of phospholipids in the cell membranes, ω3 fattyacids modify enzymes, receptors, and other proteins (Simopoulos et al.

1991). Additional studies suggest that the incorporation of ω3 fattyacids by cell membranes is enhanced in the presence of olive oil and linseed oil, emphasizing once again the importance of nutrient interactions (Cleland et

al. 1991). Cyclosporin is used widely in organ transplantation and in many

individuals its use leads to impairments in renal function and increased thromboxane formation. It was noted that the use of fish oil instead of olive oil as the vehicle for its administration in rats led to attenuation of the cyclosporine nephrotoxicity (Elzinga et al. 1987) without affecting thromboxane synthesis (Walker et al. 1989).

There is much scope for research in finding out new combinations of drugs with ω3 FA and delving deep into the causes for the interactions that happen in the body. Research in these aspects is still in budding stage.

Fishery biology, fishing techniques, taxonomy, size distribution, nutrient

value and processing of Sardines have been extensively worked out in

research institutions like Centre Marine Fisheries Research Institute and

Central Institute of Fisheries Technology. However, there have been hardly

any investigations relating to the industrial applications of this fish

commodity despite being cheap and available round the year. Hence, the

present work attempts to fill this gap.

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24

1.6 Research Objectives

The present study revolves primarily around 3 specific intentions.

a) Explicate the importance of Sardines found in the west coast of India.

b) Examine the preferential bioactivity of EPA and DHA.

c) Evaluate the pharmaceutical applications of marine PUFA from widely available sources.

As the fattyacid profile of sardines is expected to change across seasons, the first target was to analyze the seasonal change in all kinds of fatty acids for the two study species. As per those results, a good extraction technique was to be standardized to obtain a substantially pure polyunsaturated fattyacid extract which can be suggested for use in clinical research. Based on these objectives, the study is crystallized into following chapters.

Chapter 2 deals with the seasonal variation of fattyacids in S longiceps and S fimbriata, with emphasis on how EPA and DHA varies across

seasons in the two species of sardines. An explanation is attempted to elucidate why the quantity of these fatty acids vary across seasons in relation to their feeding habits.

Chapter 3 elaborates the extraction procedure of polyunsaturated fattyacids

from

S longiceps and S fimbriata and compares on how this

particular procedure fares with other known procedures for

extracting polyunsaturated fatty acids

References

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